K. E. Hekman1, C. He2, J. A. Wertheim1 1Feinberg School Of Medicine – Northwestern University,Department Of Surgery,Chicago, IL, USA 2Feinberg School Of Medicine – Northwestern University,Department Of Cell And Molecular Biology,Chicago, IL, USA
Introduction:
Vascular tissue derived from patient-specific induced pluripotent stem cells (iPSCs) suffers from premature replicative senescence, creating a significant barrier to the advancement of organ and tissue bio-engineering. Viral overexpression of longevity gene sirtuin1 as well as nutrient starvation each attenuate this premature senescence. Both are known mediators of autophagy, which is the process of cellular self-digestion that recycles intracellular components. Selective autophagy of the mitochondria, i.e., mitophagy, regulates accumulation of dysfunctional mitochondria which contribute to endothelial cell (EC) senescence. The mechanisms of premature senescence in reprogrammed iPSC-derived ECs are currently unknown.
Methods:
The iPSC lines ACS1028 and Y6 were subjected to directed differentiation and acquired endothelial lineage markers over 6 days. Cells were then purified by positive selection for VE-cadherin. Autophagy activity was monitored by quantifying the key autophagy protein, microtubule-associated proteins 1A/1B light chain 3B (LC3), through Western blotting. Mitochondrial mass, morphology and membrane potential were evaluated with MitoTracker staining. Endothelial cell function was assessed through light microscopy and quantification of nitric oxide production using the 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate substrate.
Results:
During directed differentiation from iPSCs to ECs, mitochondrial morphology evolved from globular to filamentous, and the membrane potential per mass decreased >50% (p=0.09 ACS1028, p<0.05 Y6). LC3 expression decreased by >40% (p<0.05 Y6), which correlated with increased mitochondrial mass on day 3. Mature iPSC-derived ECs had minimal autophagy activity within 5 days after purification, over which time nitric oxide production also declined by 60% (p<0.05). These iPSC-derived ECs senesced by day 13 after purification.
Conclusion:
The evolution of mitochondrial morphology and membrane potential represent remodeling during EC differentiation from iPSCs. The inverse correlation between mitochondrial mass and autophagy activity suggests this process is mediated by mitophagy. Mature iPSC-derived ECs demonstrate a significant decline in autophagy activity that correlates with the loss of mature EC function, evidenced by decreased nitric oxide synthesis, which precedes the onset of premature replicative senescence. This supports the role of mitophagy in mediating cellular senescence of iPSC-derived ECs, and renders autophagy induction a target for attenuating senescence. Overcoming this premature senescence would enable a more durable supply of patient-specific ECs for diagnostic and therapeutic applications in a wide range of disease states.
